Inhibition of Breast Cancer and Leukemia Cell-Induced Endothelial Angiogenesis by Low Molecular Weight Heparins (LMWH).

Background: Clinical studies have suggested a survival advantage associated with LMWH administration in cancer patients. Angiogenesis is an important step in tumor progression and it has been demonstrated that LMWH can affect in-vitro vessel growth induced by purified proangiogenic factors.

Objective: In this study we investigated whether two different LMWHs, enoxaparin (ENX) and dalteparin (DLT), and unfractionated heparin (UFH), were able to counteract the tube formation capacity of human microvascular endothelial cells (HMEC-1) induced by tumor cell conditioned media (TCM) or by purified factors (i.e. VEGF, bFGF, TNF-alpha and IL-1beta).

Methods: TCM were obtained after a 24h growth from three breast cancer cell lines, i.e. MCF-7, ZR75.1 and MDA.MB.231, and one human promyelocytic leukemia cell line, i.e. NB4 cells. HMEC-1 cells were resuspended in TCM or purified cytokine solutions in the presence or absence of 10 IU/ml heparins (DLT, ENX or UFH) and then seeded in a 96-well plate pre-coated with growth factor-reduced Matrigel. After 24h, tube formation was examined under phase-contrast microscopy and tube length determined by image analysis software. To characterize TCM cytokine contents, TCM were first screened for their VEGF, bFGF, TNF-alpha and IL-1beta levels by ELISA, and then inhibition studies were performed by pre-incubating TCM with specific neutralizing antibodies directed against each of these factors.

Results: TCM from all 4 tumor cell lines induced a significant (p<0.05) increase in total tube length (60 to 95% mean increase) compared with control TCM. Purified proangiogenic factors significantly (p<0.05) promoted tube formation (46 to 68% mean increase), although to a lesser extent than TCM. Angiogenesis induced by each TCM was significantly affected (76 to 100% mean inhibition) by ENX and DLT, but was less sensitive to UFH. A similar pattern of inhibition was observed with tube formation induced by purified angiogenic factors, particularly the angiogenesis induced by VEGF, bFGF, TNF-alpha or IL-1b was 100% inhibited by ENX and 65–75% by DLT, whereas minor or no inhibition was observed with UFH (VEGF= 27%; bFGF = 0%; TNF-alpha= 53%; IL-1beta= 53%). Inhibition studies with neutralizing MoAbs against proangiogenic factors demonstrated that anti-VEGF MoAb was the most effective in inhibiting tube formation induced by both breast cancer and leukemic cells (77 to 84% mean inhibition).

Conclusion: Our data indicate that heparins can inhibit the proangiogenic stimulus of both tumor cells and purified angiogenic factors on microvascular endothelium, and that the LMWHs are more effective than UFH in this experimental system.